Lentiviral vectors allowing RNAi mediated inhibition of GFAP and vimentin expression

ABSTRACT

The present invention relates to method for preventing, treating or alleviating a central nervous system (CNS) disorder using a non replicative lentivirus comprising a lentiviral genome comprising a nucleic acid sequence producing at least one functional miRNA, at least one functional shRNA and/or at least one functional siRNA, preferably derived from said shRNA, said miRNA, shRNA or siRNA being designed to silence the expression of a gene that encodes a protein of the astrocyte cytoskeleton. The present invention further relates to compositions and kits comprising such a lentivirus as well as to uses thereof.

FIELD OF THE INVENTION

The present invention relates to methods of inhibiting the expression ofa gene encoding a protein of the astrocyte cytoskeleton, said proteinbeing at least partly responsible for the formation of a glial scaroccurring in certain conditions where the Central Nervous System (CNS)is damaged. Generally, the present methods involve the use of alentiviral vector comprising a lentiviral genome comprising a nucleicacid sequence producing at least one functional micro RNA (miRNA), atleast one functional short-hairpin RNA (shRNA) and/or at least onefunctional siRNA, preferably derived from said shRNA, said miRNA, shRNAand siRNA being designed to silence the expression of a gene thatencodes a protein of the astrocyte cytoskeleton, in order to favouraxonal regeneration.

BACKGROUND

The axonal regeneration of injured and/or deteriorated neurons of thecentral nervous system constitutes a major stake in the elaboration oftherapies.

The limited capacity of the adult CNS neurons to regenerate is, inparticular, associated with the installation of a non permissivecellular environment, hostile to such a regeneration (Yiu et al., 2006,Fawcett et al., 2006, Fawcett and Asher 1999). Two types of events areresponsible for the installation of this hostile environment: productionof myelin-associated inhibitory factors, such as Nogo, MAG(myelin-associated glycoprotein) and OMgp (oligodendrocyte myelinglycoprotein) proteins, resulting from the degradation of the myelinsheath and of the accompanying oligodendrocytes, and formation of aglial scar essentially made of reactive astrocytes secreting inhibitoryproteoglycans.

The glial scar, in particular, constitutes a major physical obstacle foraxonal regeneration in conditions where the Central Nervous System (CNS)is damaged (Fawcett et al., 1999). The glial scar is mainly theconsequence of astrocytes reactivity, a phenomenon resulting inastrocytes hyperplasia and hypertrophy.

Astrocytes reactivity is characterized, on the biochemical level, by thesurexpression of at least two proteins of the astrocyte cytoskeleton,the glial fibrillary acidic protein (GFAP) and Vimentin, which arebiochemical hallmarks of the hypertrophy of the reactive astrocytes.

Matrix proteoglycans, and more specifically chondroitin sulphateproteoglycans (CSPG), are other essential elements of the glial scar,which are synthesized by different types of glial cells during reactivegliosis (Silver and Miller, 2004). Recently, a strategy was developed toimprove axonal regeneration through disintegration of CSPG thanks to aspecific enzyme, chondroitinase ABC, which was injected directly intothe scar tissue. This enzyme separates glycosaminoglycans from theprotein core of CSPG and thus eliminates their negative influence onaxonal regrowth (Bradbury et al, 2002, Moon et al, 2001). However,chondroitinase cannot be used as a therapeutic tool due to its highintrinsic toxicity for other cell components and due to its poorstability in time and space.

Other proteins of the intracellular matrix or of the cell surface arealso involved into cellular interactions, and namely axon/gliarelationships. One independent approach was that of Rutishauser, whoover expressed the sialic acid component (PSA) which, when associated tothe N-Cam protein, improves the plasticity of regenerating axons (ElMaalouf et al, 2006). This condition mimics that of the foetalenvironment, where most of N-Cam is polysialylated. No improvement offunction in animal models has however been reported to date using viralvectors overexpressing PSA.

Several myelin-associated molecules have been identified, as explainedpreviously, as inhibitors of axonal regrowth.

The most studied is Nogo, which has been identified as such thanks to anantibody named IN-1, which was found to neutralize the inhibitionprovided in vitro by myelin on axonal elongation (Caroni and Schwab,1988). Later-on this antibody was found to induce some regeneration ofthe corticospinal tract after a surgical section in adult rats (Schnelland Schwab, 1990, Brosamle et al, 2000). In this model, some recovery ofmotor functions was later described (Bregman et al, 1995). Since then,several groups attempted to generate transgenic animals with thedeletion of the genes coding for the Nogo receptor or for the protein.The conclusions regarding axonal regeneration were contradictory fromone author to another (Kim et al, 2003, Simoen et al, 2003, Zheng et al,2003). Schwab has since extended his study using a NogoA antibody(Leibscher et al, 2005, Freund et al, 2006, 2007), and a phase 1clinical trial has been launched recently using said antibody. Nogoantibodies are however associated with strong risks of immune reactions.

The other identified Myelin-associated inhibitors, MAG(myelin-associated glycoprotein) and OMpg (oligodendrocyte myelinglycoprotein), apparently share a common receptor with Nogo, semaphorin4D and ephrin B3 (Yiu et al, 2006, Fawcett et al, 2006).

A mouse knocked out for the gene encoding MAG failed to show any axonalregeneration. (Bartsch et al, 1995).

Works performed until now focused on the identification of the differentfactors responsible for the glial environment induced inhibition ofaxonal regeneration, and on the understanding of each of said factorsrespective effects in this mechanism. Therapeutic strategies implyingChondroitinase ABC, anti Nogo antibodies, PSA or antiproliferative agent(purine analog), such as Ribavirin (Pekovic et al, 2005), have beensuggested but each found associated to undesirable effects.

In order to more specifically inhibit the elements responsible for thehypertrophic part of astrocytes reactivity, transgenic mice weregenerated in which the genes coding for GFAP and vimentin wereknocked-out. These knocked-out (KO) mice were first used to develop invitro models of co-culture of wild type foetal neurons with transgenicreactive astrocytes, in order to appreciate, in a simplified system, theinfluence of the absence of these two proteins on neuron survival andneurite extension. The absence of GFAP alone, or of both proteins,allowed an increased neuronal survival and a profuse neurite extension.In addition, the expression of several proteoglycans on the surface oftransgenic astrocytes was found to mimic that of immature astrocytes(the so called radial glia) which serve as a substrate and guideimmature neurons during embryogenesis. Thus, GFAP appeared as a keyprotein in the control of astrocytes reactivity (Menet et al, 2001).These animals were then used to analyze the possible influence of theabsence of these proteins on actual axonal regeneration in a model ofspinal cord lesion. Control and mutant mice underwent a lateralhemisection of the spinal cord which induced a total paralysis of thehind limb on the lesion side. When compared with controls, double KOmice presented a reduction of the glial scar (which was apparent as soonas three days after the lesion), and a substantial regeneration, ontheir specific targets, of two systems of axons projecting respectivelyfrom the cerebral cortex and the brain stem (which was apparent aftertwo weeks). Interestingly, when challenged with a motor task (gridwalk), transgenic mice improved significantly their scores, after twoweeks, whereas controls tended to deteriorate further (Menet et al,2003).

There is an obvious need for a safe and efficient therapeutic strategy,and in particular for new tools that are able to achieve effective andspecific inhibition of undesirable expressions to thereby promote axonalregeneration, in the context of a CNS disorder, without beingcompromised by serious unwanted side effects. Such tools andprophylactic or therapeutic methods are the subject of the invention.

SUMMARY OF THE INVENTION

Inventors have now discovered that it is possible to safely, efficientlyand stably silence genes that encode factors inducing formation of aglial scar, and thereby promote axonal regeneration in the context of aCNS disorder.

The present invention relates to compounds, compositions, and methodsuseful for modulating the expression and activity of protein of theastrocyte cytoskeleton by RNA interference (RNAi) using small nucleicacid molecules, such as short interfering RNA (siRNA), as specified inthe attached claims, incorporated herein by reference.

It is indeed herein demonstrated that RNA interference (RNAi)constitutes a powerful tool to efficiently and specifically silence, onthe post-transcriptional level, the expression of a protein of theastrocyte cytoskeleton. Herein described are lentiviral vectors designedto reach this aim in a safe and controlled manner.

The present disclosure in particular provides a non replicativelentivirus comprising a lentiviral genome comprising a nucleic acidsequence producing at least one functional miRNA, at least onefunctional short-hairpin RNA (shRNA) and/or at least one functionalsiRNA, preferably derived from said shRNA, said miRNA, shRNA and siRNAbeing designed to silence the expression of a gene that encodes aprotein of the astrocyte cytoskeleton, said lentivirus being pseudotypedfor the selective transfer of the lentiviral genome into cells of thecentral nervous system, in particular in glial cells, preferably inastrocytes.

The disclosure also provides compositions, in particular pharmaceuticalcompositions comprising one or more of the present lentiviruses and apharmaceutically acceptable carrier or excipient.

Also provided are methods of preventing, treating or alleviating acentral nervous system (CNS) disorder in an animal subject, preferably ahuman, wherein glial scar is believed to play a role in the pathogenesisof the disorder, said methods comprising administering to said subject apharmaceutical composition comprising a defective lentivirus asmentioned previously, and a pharmaceutically acceptable carrier orexcipient.

In another aspect, the present disclosure provides kits comprising anyone or more of the herein-described lentivirus or compositions.Typically, the kit also comprises instructions for using the lentivirusor compositions according to the present methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RNA interference pathways (from Dykxhoorn et al. 2003)

-   -   a) Structure of a short interfering RNA (siRNA). Molecular of an        siRNA include 5′ phosphorylated ends, a 19 to 22 nucleotides        duplexed region and 2 nucleotides unpaired and unphosphorylated        3′ ends that are characteristic of RNAse III cleavage products.    -   b) siRNA pathway. In an initiation phase, double stranded RNA        (dsRNA) is cleaved by the enzyme DICER, giving rise to siRNA        duplex. In an activation phase, the siRNA duplex is associated        to the enzymatic complex that leads to recognition of the mRNA        target and enzymatic degradation of the mRNA target.    -   c) miRNA pathway. DICER also cleaves the ˜70 nucleotides hairpin        miRNA precursor to produce ˜22 nucleotides miRNA.

FIG. 2: Methods to generate siRNAs in vivo from plasmidic or viralvectors

-   -   A) sense and antisense strands of the siRNA are expressed from        tandem polIII promoters    -   B) short hairpin RNA (shRNA) is expressed from a singe polIII        promoter    -   C) Imperfect duplex hairpin, based on pre-miRNA is expressed        from polII promoter and is processed by DICER into a mature        miRNA

FIG. 3: Screening of shRNAs targeting GFAP and Vimentin—Analysis ofGFAP-GFP and Vimentin-GFP expression by Fluorescent Activated CellSorting (FACS), 72 h after cotransfection

-   -   A) Percentages of GFP positives cells were measured for the        different shGFAP contructs    -   B) Mean fluorescence intensity was measured for the different        shGFAP contructs    -   C) Percentages of GFP positives cells were measured for the        different shVIM constructs    -   D) Mean fluorescence intensity was measured for the different        shVIM contructs

FIG. 4: Design of a lentiviral vector allowing expression of shGFAP orshVIM. LTR, ψ, and Flap are HIV-1 derived sequences (the LTRs, thepackaging sequence, and the central Flap element, respectively). P_(U6)and P_(PGK) are respectively the human U6 promoter and the ubiquitousPGK promoter. GFP is the coding sequence of Green Fluorescent Protein,and WPRE, the Woodchuck hepatitis virus responsive element.

FIG. 5: Lentiviral mediated silencing of GFAP and Vimentin expression byRNAi in primary astrocytes. Astrocytes were transduced with variousamounts of vectors, expressed in number of viral particles per seededcell (MOI=multiplicity of infection).

FIG. 6: Effects of the lentiviral vectors on neuronal survival andneurite growth.

-   -   A) Neuronal survival was assayed by counting the βIII-tubulin        positive cells.    -   B) Neurite outgrowth was evaluated by measuring the surface        occupied by perikarya and neurites per neuron.        -   NT=non transduced cells. Lv-PGK-GFP, Lv-shRANDOM and Lv-shG1            are used as control vectors (*p<0.05; **p<0.01; ***p<0.001;            Mann-Whitney test).

FIG. 7: Schematic representation of the intraparenchymal injection ofthe Lv-shGFAP and Lv-shVIM lentiviral vectors

-   -   A) Representation of the injections sub-sites    -   B) Representation of the injections sites

FIG. 8: In vivo inhibition of GFAP expression by the lentiviral vectorLv-shGFAP. These pictures presente GFP and GFAP immunostaining on spinalcord frozen sections of a previously hemisected mouse. The transducedarea is visualized by GFP immunostaining (green) and GFAP immunostaining(red).

A) and B): Two weeks after transduction, GFAP expression is decreased inspinal cord transduced with Lv-shGFAP (A) when compared with the controlLv-PGK-GFP vector (B).C) and D): Five weeks after transduction, GFAP expression is decreasedin spinal cord transduced with Lv-shGFAP (C) when compared with thecontrol Lv-PGK-GFP vector (D).

FIG. 9: In vivo inhibition of Vimentin expression by the lentiviralvector Lv-shVIM. These pictures presente GFP and VIM immunostaining onspinal cord frozen sections of a previously hemisected mouse. Thetransduced area is visualized by GFP immunostaining (green) and GFAPimmunostaining (red).

A) and B): five weeks after transduction, Vimentin expression isdecreased in spinal cord transduced with Lv-shVIM (A) when compared withthe control Lv-shRANDOM vector (B).

FIG. 10: Effects of the Lv-shGFAP and Lv-shVIM vectors on functionalrecovery in an animal model of spinal cord injury.

The different vectors were injected in mice in which inventors haveperformed a total unilateral hemisection of the spinal cord. Grid runwaytest was performed 5 weeks after lesion and injection of the lentiviralvectors. PBS, Lv-PGK-GFP and Lv-H1-shRANDOM are used as controls.Recuperation was measured by the difference between faults number at 2weeks and faults number at five weeks. Statistical significancy wasevaluated by non-parametric Mann-Whitney test (*p<0.05)

FIG. 11: Analysis of serotonergic regrowth in the ventral horn afterhemisection and injection of the Lv-shGFAP and Lv-shVIM vectors. % ofthe surface occupied by serotonergic fibres in the ventral horn of thelesioned side was evaluated for each vectors condition in comparisonwith the non-lesioned side.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In describing, exemplifying and claiming the present invention, thefollowing terminology will be used in accordance with the definitionsset out below. Where not otherwise indicated, the terms are intended tohave the meaning generally recognized in the art.

By “GFAP” is meant GFAP peptide or protein or a naturally occurringfragment thereof, wherein the peptide or protein is encoded by the GFAPgene.

By “vimentin” is meant vimentin peptide or protein or a naturallyoccurring fragment thereof, wherein the peptide or protein is encoded bythe vimentin gene.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as comprising non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides.

The term “short interfering nucleic acid”, “siRNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, “miRNA”, “micro RNA” as usedherein refers to any nucleic acid molecule capable of mediating RNAinterference “RNAi” or gene silencing in a sequence-specific manner. RNAinterference (RNAi) describes a process wherein double-stranded RNA(dsRNA), when present inside a cell, inhibits expression of anendogenous gene that has an identical or nearly identical sequence tothat of the dsRNA. Inhibition is caused by the specific degradation ofthe messenger RNA (mRNA) transcribed from the target gene. In greaterdetail, RNA interference describes a process of sequence-specificpost-transcriptional gene silencing in animals mediated by theexpression of “short interfering RNAs” (siRNAs) after in situ cleavage(Brummelkamp et al., 2002). The initial basic process involves doublestranded RNA (dsRNA) that is/are processed by cleavage into shorterunits (the so called siRNA) that guide recognition and targeted cleavageof homologous target messenger RNA (mRNA) (see FIG. 1 a).

The method does not require the time-consuming genetic manipulationsneeded for classical gene knock-out strategies and has therefore emergedas a valuable tool in molecular genetics that may also be applied tohuman therapy.

The currently known mechanism of RNAi can be described as follows:

The processing of dsRNA into siRNAs, which in turn induces degradationof the intended target mRNA, is a two-step RNA degradation process. Thefirst step involves a dsRNA endonuclease (ribonuclease III-like; RNaseIII-like) activity that processes dsRNA into smaller sense and antisenseRNAs which are most often in the range of 21 to 25 nucleotides (nt)long, giving rise to the so called short interfering RNAs (siRNAs). ThisRNase III-type protein is termed “Dicer”. In a second step, theantisense siRNAs produced combine with, and serve as guides for, adifferent ribonuclease complex called RNA-induced silencing complex(RISC), which allows annealing of the siRNA and the homologoussingle-stranded target mRNA, and the cleavage of the target homologoussingle-stranded mRNAs. Cleavage of the target mRNA has been observed toplace in the middle of the duplex region complementary to the antisensestrand of the siRNA duplex and the intended target mRNA (see FIG. 1 b)(Dykxhoorn et al., 2003 for review).

Micro RNAs (miRNAs) constitute non coding RNAs of 21 to 25 nucleotides,which controls genes expression at post-transcriptional level. miRNAsare synthesized from ARN polymerase II or ARN polymerase III in apre-miRna of 125 nucleotides. Pre-miRNA are cleaved in the nucleus bythe enzyme Drosha, giving rise to a precursor called imperfect duplexhairpin RNA (or miRNA-based hairpin RNA). These imperfect duplex hairpinRNAs are exported from the nucleus to the cytoplasm by exportin-5protein, where it is cleaved by the enzyme DICER, giving rise to maturemiRNAs. miRNAs combine with RISC complex which allows total or partialannealing with the homologous single-stranded target mRNA. Partialannealing with the mRNA leads to the repression of protein translation,whereas total annealing leads to cleavage of the single-stranded mRNA(see FIG. 1 c). (Dykxhoorn et al., 2003 for review).

At least three methods to generate RNAi-mediated gene silencing in vivoare known and usable in the context of the present invention (Dykxhoornet al., 2003 for review): siRNAs with a single sequence specificity canbe expressed in vivo from plasmidic or viral vectors using:

-   -   Tandem polymerase III promoter that express individual sense and        antisense strands of the siRNAs that associate in trans (see        figure A3-A)    -   a single polymerase III promoter that expresses short hairpin        RNAs (shRNAs) (see figure A3-B)    -   a single polymerase II promoter that expresses an imperfect        duplex hairpin RNA (pre-miRNA) which is processed by DICER        giving rise to a mature miRNA (see figure A3-C).

By “antisense strand” is meant a nucleotide sequence of a siRNA moleculehaving complementarity to a sense region of the siRNA molecule. Inaddition, the antisense strand of a siRNA molecule comprises a nucleicacid sequence having homology with a target nucleic acid sequence.

By “sense strand” is meant a nucleotide sequence of a siRNA moleculehaving complementarity to an antisense region of the siRNA molecule.

By “modulate” and “modulation” is meant that the expression of the gene,or level of RNA molecule or equivalent RNA molecules encoding one ormore proteins or protein subunits, or activity of one or more proteinsor protein subunits is up regulated or down regulated, such thatexpression, level, or activity is greater than or less than thatobserved in the absence of the modulator. For example, the term“modulate” can mean “inhibit” and within the scope of the invention, thepreferred form of modulation is inhibition but the use of the word“modulate” is not limited to this definition.

By “inhibit”, “silence” or “down regulate” it is meant that the levelsof expression product or level of RNAs or equivalent RNAs encoding oneor more gene products is reduced below that observed in the absence ofthe nucleic acid molecule of the invention. In one embodiment,inhibition with a nucleic acid molecule capable of mediating RNAinterference (siRNA, shRNA, miRNA) preferably is below that levelobserved in the presence of an inactive or attenuated molecule that isunable to mediate an RNAi response.

By “target protein” is meant any protein whose expression or activity isto be modulated. Preferred target proteins are GFAP and vimentine.

By “target nucleic acid” or “target gene” is meant any nucleic acidsequence whose expression or activity is to be modulated. The targetnucleic acid can be DNA or RNA. In the context of the invention, the“target gene” is a gene that encodes a protein of the astrocytecytoskeleton, typically the GFAP or the vimentine gene.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. The subject may be a non-human animal, preferably amammal. The preferred subject is a human subject.

Glial fibrillary acidic protein (GFAP) is a 55 kDa cytosolic proteinthat is a major structural component of astroglial filaments and is themajor intermediate filament protein in astrocytes. GFAP is specific toastrocytes. This protein helps to maintain astrocyte mechanical strengthand shape. This protein is involved in reactive astrocyte hypertrophy.

Vimentin is a 57 kDa cytosolic protein that is a major structuralcomponent of astroglial filaments and a major intermediate filamentprotein in astrocytes. Vimentin is specifically re-expressed in reactiveastrocytes after CNS injury. This protein is involved in reactiveastrocyte hypertrophy.

New Approach for Axonal Regeneration:

The brain contains two major types of cells: neurons and glial cells.Neurons are the most important cells in the brain, and are responsiblefor maintaining communication within the brain via electrical andchemical signalling. Glial cells function mainly as structuralcomponents of the brain, and they are approximately 10 times moreabundant than neurons. Glial cells of the central nervous system (CNS)are astrocytes and oligodendrocytes.

Astroglial cells respond to trauma and ischemia with reactive gliosis(also called “astrocytic activation”), a reaction characterized byincreased astrocytic proliferation and hypertrophy. Although beneficialto a certain extent, excessive gliosis may be detrimental, contributingto neuronal death in neurodegenerative diseases and in SNC trauma.

Astrocytic activation evidenced by increased glial fibrillary acidicprotein has been found for example in multiple sclerosis (Malmestrom C,Haghighi S, Rosengren L, Andersen O, Lycke J: Neurofilament lightprotein and glial fibrillary acidic protein as biological markers in MS.Neurology 2003; 61:1720-1725), temporal lobe epilepsy (Briellmann R S,Kalnins R M, Berkovic S F, Jackson G D: Hippocampal pathology inrefractory temporal lobe epilepsy: T2-weighted signal change reflectsdentate gliosis. Neurology 2002; 58:265-271), amyotrophic lateralsclerosis (Lexianu M, Kozovska M, Appel S H: Immune reactivity in amouse model of familial ALS correlates with disease progression.Neurology 2001; 57:1282-1289), systemic lupus erythematosus (Trysberg E,Nylen K, Rosengren L E, Tarkowski A: Neuronal and astrocytic damage insystemic lupus erythematosus patients with central nervous systeminvolvement. Arthritis Rheum 2003; 48:2881-2887), human immunodeficiencyvirus dementia, Alzheimer's dementia, traumatic injury, Parkinsondisease (Teismann and Schulz, 2004) and Alzheimer's disease (Sjobeck andEnglund, 2003).

The present disclosure provides a novel strategy of axonal regenerationbased on the neutralization, via gene transfer, of the elements which,during reactive astrocytic gliosis, are responsible for the formation ofa biochemical and physical barrier, the so called “glial scar”, composedof reactive astrocytes and proteoglycans. The biochemical hallmark ofastrogliosis is the massive upregulation of the intermediate filamentproteins (IF) GFAP and vimentin.

Inventors herein demonstrate that said proteins of the astrocytecytoskeleton are appropriate and valuable targets in the context oftherapy, in particular of human therapy. Inventors herein providevectors carrying nucleic acid molecule mediating RNAi, in particularnucleic acid molecules producing miRNA, shRNA and/or siRNA moleculesthat down regulate expression of proteins of the astrocyte cytoskeletonby RNA interference. The inventors herein demonstrate the beneficialimpact of these vectors on health in several animal models of CNSdisorder. The vectors were shown to be effective in vitro, as well as invivo in animal models (see experimental part of the presentapplication).

The vectors benefit from the technology of ribonucleic acid interference(RNAi), which is described above in great details.

Employing siRNAs in living animals, especially humans, was a challenge,since siRNAs show different effectiveness in different cell types in amanner yet poorly understood: some cells respond well to siRNAs and showa robust knockdown, others show no such knockdown (even despiteefficient transfection). However it was a successful approach, whichproved to be both safe and very efficient.

Nucleic Acid Molecules Capable of Mediating RNA Interference

Preferred molecules capable of mediating RNA interference advantageouslydown regulate at least 60%, preferably at least 70%, preferably at least80%, even more preferably at least 90%, of the target proteinexpression.

Preferred shRNA designed to silence a gene encoding GFAP are identifiedbelow (sequences in black design the siRNA sequence produced aftercleavage of the shRNA by DICER):

Murine genome targeting sequence SEQ ID NO 1: (SEQ ID NO: 1)ACCGAGAGAGATTCGCACTCAATATTCAAGAGATATTGAGTGCGAATCTC TCTCTTTTTATCGATG;Human, murine and rat genome targeting sequence SEQ ID NO: 2: (SEQ IDNO: 2) ACCGAGATCGCCACCTACAGGAAATTCAAGAGATTTCCTGTAGGTGGCGATCTCTTTTTATCGATG. The siRNA GAGAGAGATTCGCACTCAATA is herein identifiedas SEQ ID NO: 3. The siRNA TATTGAGTGCGAATCTCTCTC is herein identified asSEQ ID NO: 4. The siRNA GAGATCGCCACCTACAGGAAA is herein identified asSEQ ID NO: 5. The siRNA TTTCCTGTAGGTGGCGATCTC is herein identified asSEQ ID NO: 6. Preferred shRNA designed to silence the murine geneencoding vimentin are: (SEQ ID NO: 7)ACCGAATGGTACAAGTCCAGGTTTGTTCAAGAGACAAACTTGGACTTGTA CCATTCTTTTTCTCGAGG.(SEQ ID NO: 8) ACCGAGAGAAATTGCAGGAGGAGATTCAAGAGATCTCCTCCTGCAATTTCTCTCTTTTTCTCGAGG The siRNA GAATGGTACAAGTCCAGGTTTG is herein identifiedas SEQ ID NO: 9. The siRNA CAAACTTGGACTTGTACCATTC is herein identifiedas SEQ ID NO: 10. The siRNA GAGAGAAATTGCAGGAGGAGA is herein identifiedas SEQ ID NO: 11. The siRNA TCTCCTCCTGCAATTTCTCTC is herein identifiedas SEQ ID NO: 12.

Preferred siRNA targeting the human gene encoding vimentin, described byHarborth et al. (2001), have been identified by inventors as usable, inthe context of the present invention, to prevent, treat or alleviate theabove mentioned disorders associated with the formation of a glial scar.The sense and antisense sequences of these siRNA molecules are indicatedbelow:

1) SEQ ID NO 13 sense sequence: GAAUGGUACAAAUCCAAGU(dTdT) SEQ ID NO 14antisense sequence: ACUUGGAUUUGUACCAUU(dTdT) 2) SEQ ID NO 15 sensesequence: AUGGAAGAGAACUUUGCCG(dTdT) SEQ ID NO 16 antisense sequence:CGGCAAAGUUCUCUUCCAU(dTdT) 3) SEQ ID NO 17 sense sequence:UACCAAGACCUGCUCAAUG(dTdT) SEQ ID NO 18 antisense sequence:CAUUGAGCAGGUCUUGGUA(dTdT)

Lentivirus Vectors

Inventors demonstrate that the above described nucleic acid molecule,capable of mediating RNA interference, can be safely, efficiently anddurably expressed in target cells by using appropriate expressionvectors herein described.

An appropriate expression vector is a non replicative lentiviruscomprising a lentiviral genome comprising a nucleic acid sequenceproducing at least one functional micro RNA (miRNA), at least onefunctional short-hairpin RNA (shRNA) and/or at least one functionalshort interfering RNA (siRNA), said siRNA being preferably derived fromsaid shRNA, said miRNA, shRNA and siRNA being designed to silence theexpression of a gene that encodes a protein of the astrocytecytoskeleton, said lentivirus being pseudotyped for the selectivetransfer of the lentiviral genome into cells of the central nervoussystem, preferably into glial cells, even more preferably intoastrocytes.

In a particular embodiment, the non replicative lentivirus of theinvention, comprises a lentiviral genome as previously described furthercomprising a second nucleic acid sequence producing at least onefunctional miRNA, at least one functional shRNA and/or at least onefunctional siRNA, preferably derived from said shRNA, said miRNA, shRNAand siRNA being designed to silence the expression of a gene encoding adifferent protein of the astrocyte cytoskeleton.

Lentiviruses are complex retroviruses capable of transducing cells whichare not mitotically active, such as cells of the nervous system, inparticular certain cell subpopulations of the central nervous system.These viruses include in particular Human Immunodeficiency Virus type 1(HIV-1), Human Immunodeficiency Virus type 2 (HIV-2), SimianImmunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV),Equine Infectious Anaemia Virus (EIAV), Bovine Immunodeficiency Virus(BIV), Visna Virus of sheep (VISNA) and Caprine Arthritis-EncephalitisVirus (CAEV). A preferred lentivirus according to the present inventionis selected in the above mentioned list of viruses.

Like other retroviruses, lentiviruses have gag, pol and env genesflanked by two LTR (Long Terminal Repeat) sequences. Each of these genesencodes many proteins which are initially expressed in the form of asingle precursor polypeptide. The gag gene encodes the internalstructural proteins (capsids and nucleocapsids). The pol gene encodesthe reverse transcriptase, the integrase and the protease. The env geneencodes the viral envelope glycoprotein and also contains a cis-actingRRE (Rev Responsive Element) responsible for exporting the viral RNA outof the nucleus. The 5′ and 3′ LTR sequences serve to promote thetranscription and the polyadenylation of the viral RNAs. The LTRcontains all the other cis-acting sequences necessary for viralreplication. Sequences necessary for the reverse transcription of thegenome (tRNA primer binding site) and for encapsidation of the viral RNAinto particles (site Ψ) are adjacent to the 5′ LTR. If the sequencesnecessary for encapsidation (or for packaging of the retroviral RNA intoinfectious virions) are absent from the viral genome, the genomic RNAwill not be actively encapsidated.

The construction of lentiviral vectors for gene transfer applicationshas been described, for example, in patents U.S. Pat. No. 5,665,577, EP386 882, U.S. Pat. No. 5,981,276 and U.S. Pat. No. 6,013,516 or else inpatent application WO 99/58701.

The vectors used in the present invention are non replicative, in otherwords they comprise a defective lentiviral genome, i.e., a genome inwhich at least one of the gag, pol and env genes has been inactivated ordeleted. These vector genomes are encapsidated in a protein particlecomposed of the structural lentiviral proteins and in particular of theenvelope glycoprotein.

The recombinant lentiviruses according to the invention are thusgenetically modified in such a way that certain genes constituting thenative infectious virus are eliminated and replaced with a nucleic acidsequence of interest to be introduced into the target cells. Afteradsorption of the virus on the cell membrane, said virus injects itsnucleic acid into the cell and, after reverse transcription, saidnucleic acid can integrate into the genome of the host cell. The geneticmaterial thus transferred is then transcribed and possibly translatedinto proteins inside the host cell. When the lentiviral vector is a nonintegrative lentiviral vector, the genetic material transferred in hostcells is present in episomal forms, named 1 LTR or 2 LTR circles.

A preferred non replicative lentivirus herein described is a lentivirusdeprived of any lentiviral coding sequence. It is also deleted of theenhancer region of the U3 region of the LTR3′. Particularly preferredlentiviral vector are pseudotyped vectors that allow targeting of a cellpopulation of the central nervous system. The term “pseudotyping”denotes a recombinant virus comprising an envelope different from thewild-type envelope. In the context of the present invention, the vectorsexpress an envelop protein which direct the vector to various cells,including the cells of the Central Nervous System.

An appropriate envelope glycoprotein is a vesiculovirus envelopeglycoprotein such as the envelope glycoprotein of the vesicularstomatitis virus (VSV). This envelope exhibits advantageouscharacteristics, such as resistance to ultracentrifugation and a verybroad tropism. Unlike other envelopes, such as those of the conventionalretroviruses (amphotropic and ecotropic MLV retroviruses or HIV gp120,but also many others), the VSV glycoprotein is not labile afterultracentrifugation. This makes it possible to concentrate the viralsupernatants and to obtain high infectious titres. Moreover, thisenvelope confers on the virions a very broad tropism, in particular invitro, allowing the infection of a very large number of cell types,including cells of the central nervous system, in particular glial cellssuch as astrocytes. The receptor for this envelope is thought to be aphosphatidylserine motif present at the surface of many cells of variousspecies. VSV-G is an example of such a VSV envelop glycoprotein.

Preferred vectors allow targeting of glial cells, preferably glial cellsof the astrocyte type. These pseudotyped viral vectors are useful forthe transfer and the expression in vitro, ex vivo and in vivo of nucleicacid sequences of interest preferentially within astrocytes.

The term “preferentially” should be understood to mean that thelentiviruses according to the invention target essentially astrocytesbut are, nevertheless, capable of transfecting other cell types, such asother glial cells of the central nervous system. Other nerve cellsubpopulations which may be targeted by vectors of the invention are,for example, microglial cells, endothelial cells or oligodendrocytes.

Preferred envelopes allowing the preferential targeting of glial cellsof the astrocyte type are lyssavirus envelopes, in particular a virusenvelop of the rabies virus serogroup selected from the group consistingof Rabies (RAB); Duvenhague (DUV), European Bat type 1 (EB-1), EuropeanBat type 2 (EB-2), Kotonkan (KOT), Lagos Bat (LB), Mokola (MOK),Obodhiang (OBD) and Rochambeau (RBU), or any chimeric composition ofthese envelopes. In a preferred embodiment, inventors use lentiviralvectors, for example of the HIV type, pseudotyped with an envelope ofthe PV (rabies virus) or MOK (Mokola virus) type. Other envelopeglycoproteins that can be used to allow preferential targeting of glialcells are alphaviruses envelopes, in particular the Ross River virus(RRV) glycoprotein, and arenaviridae envelopes, in particular theLymphocytic choriomeningitis virus (LCMV) glycoprotein.

In a particular embodiment, the lentivirus comprises a lentiviral genomecomprising, between wild type or modified (att mutants) LTR5′ and LTR3′sequences, a Psi (Ψ) encapsidation sequence, at least one codingsequence producing at least one functional miRNA, at least onefunctional shRNA, and/or at least one functional siRNA preferablyderived from said shRNA, and optionally: a promoter, a sequenceenhancing RNA nuclear import such as the cppT-CTS, a sequence enhancingRNA nuclear export, a transcription regulation element, and/or a mutatedintegrase.

The above mentioned promoter can be a viral or a cellular promoter.

A preferred cellular promoter usable, in the context of the presentinvention, to express a shRNA, may be selected from the group consistingof U6, H1 and 7SK RNA polymerase III promoter.

A preferred viral promoter usable, in the context of the presentinvention, to express a miRNA targeting GFAP or vimentine, may beselected from the group consisting of CMV, TK, RSV LTR polymerase IIpromoter.

A preferred cellular promoter usable, in the context of the presentinvention, to express a miRNA targeting GFAP or vimentine, may beselected from the group consisting of PGK, Rho, EF1α, GFAP, Vimentin,Nestin, S100β.

In a particular embodiment, the promoter is a transactivator inducedpromoter as further explained below, preferably comprising a pluralityof transactivator binding sequences operatively linked to the nucleicacid sequence producing shRNA.

A particularly preferred sequence enhancing the RNA nuclear import isthe lentiviral cPPT CTS (flap) sequence from HIV-1. Other sequences,usable in the context of the present invention, enhancing RNA nuclearimport are lentiviral cPPT CTS sequences from (HIV-2, SIV, FIV, EIAV,BIV, VISNA and CAEV). A particularly preferred sequence enhancing theRNA nuclear export is a sequence comprising the HIV-1 REV responseelement (RRE) sequence. Another sequence, usable in the context of thepresent invention, which enhances the RNA nuclear export, is the CTEsequence (Oh et al 2007). Preferred posttranscriptional regulationelements may be selected from Woodchuck hepatitis virus responsiveelement (WPRE), APP UTR5′ region and TAU UTR3′. A preferred regulationelement will be an insulator sequence selected from the group consistingof, for example, MAR, SAR, S/MAR, scs and scs'.

A preferred lentivirus is non integrative (EP 1761635). Such alentivirus comprises a mutated integrase in order to limit the risk ofinsertion mutagenesis. Preferably the integrase comprises a mutation inat least one of its basic region (N, L and/or Q regions, preferably Land Q regions) and/or catalytic region. The lentivirus integration canalso be silenced by mutating the att sequence of the LTRs, by mutatingthe CA motif of the att sequence (Nightinghale et al. 2006).

The lentiviral vectors according to the invention can be prepared invarious ways, notably by transient transfection(s) into producer cells(or using stable producer cell lines) and/or by means of helper viruses.

The method according to the invention comprises, according to aparticularly preferred embodiment, the transfection of a combination ofa minimum of three plasmids in order to produce a recombinant virion ora recombinant retrovirus.

A first plasmid provides the lentiviral vector genome comprising thecis-acting viral sequences necessary for the correct functioning of theviral cycle. Such sequences include preferably one or more lentiviralLTRs, a Psi (ψ) packaging sequence, reverse transcription signals, apromoter and/or an enhancer and/or polyadenylation sequences. In thisvector, the LTRs can also be modified so as to improve the expression ofthe transgene or the safety of the vector. Thus, it is possible tomodify, for example, the sequence of the 3′ LTR by eliminating the U3region [modified sequence herein identified as LTR(ΔU3)] (see WO99/31251). One can also introduce the transgene cassette(promoter+transgene) in the vectors genome between the LTRs, or in placeof the U3 region of the LTR 3′.

According to a particular embodiment of the invention, it is a vectorplasmid comprising a recombinant lentiviral genome of sequenceLTR-psi-Promoter-transgene-LTR which allows expression of the vector RNAwhich will be encapsidated in the virions.

A preferred vector plasmid comprises a recombinant lentiviral genome ofsequence LTR-psi-flap-Promoter-transgene-LTR wherein flap designates thesequence cPPT CTS enhancing the ARN nuclear import.

Another preferred vector plasmid comprises a recombinant lentiviralgenome of sequence LTR-psi-flap-Promoter-transgene-WPRE-LTR, whereinflap designates the sequence cPPT CTS which improves the transduction ofnon dividing cells, and in particular which enhances the ARN nuclearimport, and wherein WPRE (Woodchuck hepatitis virus responsive element)is a transcription regulation element, advantageously used to enhancethe transgene expression level.

In the present invention, the transgene or nucleic acid of interestproduces at least one functional nucleic acid molecule capable ofmediating RNA interference, preferably at least one functional miRNA, atleast one functional short-hairpin RNA (shRNA), or at least onefunctional siRNA derived from said shRNA, said nucleic acid moleculebeing designed to silence the expression of at least one target gene, inparticular a gene that encodes a protein of the astrocyte cytoskeleton,said protein being selected preferably, from GFAP and vimentin.

The transgene is typically placed under the control of a transcriptionalpromoter. A promoter that is particularly useful in the context of thepresent invention has a transcription machinery that is compatible withmammalian genes, can be compatible with expression of genes from a widevariety of species, preferably has a high basal transcription rate,recognizes termination sites with a high level of accuracy. A preferredpromoter will preferably be sufficient to direct the transcription of adistally located sequence, which is a sequence linked to the 3′ end ofthe promoter sequence in a cell.

Since long poly A tails compromise the silencing effect of shRNAs, theirexpression is appropriately driven by RNA polymerase III whichrecognizes a run of 5T residues as a stop signal and does not thereforerequire a poly A sequence to terminate transcription.

Suitable promoters include, for example, RNA polymerase (pol) IIIpromoters including, but not limited to, the (human and murine) U6promoters, the (human and murine) H1 promoters, and the (human andmurine) 7SK promoters. In addition, a hybrid promoter also can beprepared that contains elements derived from, for example, distincttypes of RNA polymerase (pol) III promoters. Modified promoters thatcontain sequence elements derived from two or more naturally occurringpromoter sequences can be combined by the skilled person to effecttranscription under a desired set of conditions or in a specificcontext. For example, the human and murine U6 RNA polymerase (pol) IIIand H1 RNA pol III promoters are well characterized and useful forpracticing the invention. One skilled in the art will be able to selectand/or modify the promoter that is most effective for the desiredapplication and cell type so as to optimize modulation of the expressionof one or more genes. The promoter sequence can be one that does notoccur in nature, so long as it functions in a eukaryotic cell,preferably a mammalian cell.

Expression of the transgene or nucleic acid of interest, here the atleast one functional miRNA, shRNA or siRNA derived from said shRNA, maybe externally controlled by treating the cell with a modulating factor,such as doxycycline, tetracycline or analogues thereof. Analogues oftetracycline are for example chlortetracycline, oxytetracycline,demethylchloro-tetracycline, methacycline, doxycycline and minocycline.Conditional suppression of genes may indeed be important for therapeuticapplications by allowing time and/or dosage control of the treatment orby permitting to terminate treatments at the onset of unwanted sideeffects.

Reversible gene silencing may be implemented using a transactivatorinduced promoter together with said transactivator. Such atransactivator induced promoter comprises control elements for theenhancement or repression of transcription of the transgene or nucleicacid of interest producing miRNA, shRNA and/or siRNA. Control elementsinclude, without limitation, operators, enhancers and promoters. Atransactivator inducible promoter, in the context of the presentinvention, is transcriptionally active when bound to a transactivator,which in turn is activated under a specific set of conditions, forexample, in the presence or in the absence of a particular combinationof chemical signals, preferably by a modulating factor selected forexample from the previous list.

The transactivator induced promoter may be any promoter herein mentionedwhich has been modified to incorporate transactivator binding sequences,such as several tet-operon sequences, for example 7 tet-operonsequences, preferably in tandem. Such sequences can for example replacethe functional recognition sites for Staf and Oct-1 in the distalsequence element (DSE) of the U6 promoter, preferably the human U6promoter.

Advantageously, the transactivator induced promoter comprises aplurality of transactivator binding sequences operatively linked to thenucleic acid sequence producing shRNAs.

The transactivator may be provided by a nucleic acid sequence, in thesame expression vector or in a different expression vector, comprising amodulating factor-dependent promoter operatively linked to a sequenceencoding the transactivator. The term “different expression vector” isintended to include any vehicle for delivery of a nucleic acid, forexample, a virus, plasmid, cosmid or transposon. Suitable promoters foruse in said nucleic acid sequence include, for example, constitutive,regulated, tissue-specific or ubiquitous promoters, which may be ofcellular, viral or synthetic origin, such as CMV, RSV, PGK, EF1α, NSE,synapsin, β-actin, GFAP.

A preferred transactivator according to the present invention is thertTA-Oct.2 transactivator composed of the DNA binding domain of rtTA2-M2and of the Oct-2^(Q)(Q→A) activation domain.

Another preferred transactivator according to the present invention isthe rtTA-Oct.3 transactivator composed of the DNA binding domain of theTet-repressor protein (E. coli) and of the Oct-2^(Q)(Q→A) activationdomain.

Both are described in patent application WO 2007/004062.

As used herein, the term “operatively linked” means that the elementsare connected in a manner such that each element can serve its intendedfunction and the elements, together can serve their intended function.In reference to elements that regulate gene expression, “operativelylinked” means that a first regulatory element or coding sequence in anucleotide sequence is located and oriented in relation to a secondregulatory element or coding sequence in the same nucleic acid so thatthe first regulatory element or coding sequence operates in its intendedmanner in relation with the second regulatory element or codingsequence.

When the lentivirus comprises a transactivator induced promoter, saidlentivirus may further advantageously comprise a WPRE which is able toenhance the expression of the transactivator.

A second plasmid, for trans-complementation, provides a nucleic acidencoding the protein products of the gag and pol lentiviral genes. Theseproteins are derived from a lentivirus and preferably originate fromHIV, in particular HIV-1. The second plasmid is devoid of encapsidationsequence, of sequence encoding an envelope and, advantageously, is alsodevoid of lentiviral LTRs. As a result, the sequences encoding gag andpol proteins are advantageously placed under control of a heterologouspromoter, for example a viral, cellular, etc. promoter, which may beconstitutive or regulated, weak or strong. It is preferably atrans-complementing plasmid comprising a sequenceCMV-Δpsi-gag-pol-Δenv-PolyA. This plasmid allows the expression of allthe proteins necessary for the formation of empty virions, except theenvelope glycoproteins. It is understood that the gag and pol genes mayalso be carried by different plasmids.

A third plasmid provides a nucleic acid which allows the production ofthe chosen envelope (env) glycoprotein. This envelope may be chosen fromthe envelopes mentioned above, in particular an envelope of arhabdovirus, more particularly of a lyssavirus, even more preferably anenvelop of the Mokola virus. This vector is preferentially devoid of alllentiviral sequences, encapsidation sequence and of sequences encodinggag or pol and, advantageously, is also devoid of lentiviral LTRs.

Advantageously, the three vectors used do not contain any homologoussequence sufficient to allow a recombination. The nucleic acids encodinggag, pol and env may advantageously be cDNAs prepared according toconventional techniques, from sequences of the viral genes available inthe prior art and on databases.

For the production of the non replicative lentiviruses, the vectorsdescribed above are introduced into competent cells and the virusesproduced are harvested. The cells used may be any competent cell,preferably mammalian cell, for example animal or human cell, which isnon pathogenic. Mention may, for example, be made of 293 cells,embryonic cells, fibroblasts, muscle cells, etc.

A preferred method for preparing a non replicative recombinantlentivirus, according to the invention, comprises transfecting apopulation of competent cells with a combination of vectors as describedabove, and recovering the viruses thus produced.

A particularly advantageous method for producing lentiviruses capable ofsilencing in vivo the expression of a gene that encodes a protein of theastrocyte cytoskeleton, in particular in human astrocytes, comprisestransfection of competent cells with:

a) a vector plasmid comprising a sequence, as described previously, suchas LTR-psi-Promoter-transgene-LTR(ΔU3),b) a trans-complementing plasmid comprising a sequenceCMV-Δpsi-gag-pol-Δenv-PolyA, andc) an envelope plasmid comprising a sequence CMV-env-PolyA, the envelopebeing preferably an envelope of the rabies virus serogroup.

The lentiviruses of the invention may also be prepared, as explainedpreviously, from an encapsidation cell line producing one or more gag,pol and env proteins.

Therapeutic Uses

The lentiviruses according to the invention may be used for preparing acomposition intended for gene transfer into astrocytes in vivo or exvivo.

The lentiviruses according to the invention may in particular be usedfor preparing a pharmaceutical composition intended to prevent, treat oralleviate a nervous system, in particular a central nervous system(CNS), disorder in an animal subject, preferably a mammal, in particulara human.

Another subject of the invention lies in the combined use of severalidentical or different lentiviruses as herein described, for the purposeof transferring and expressing several identical or different miRNAs,shRNAs and/or siRNA in the cells of the nervous system, in particular inglial cells, preferably in astrocytes. The combined use may comprisesequential administrations of the various viruses, or a simultaneousadministration.

The lentiviruses of the invention may further allow the transport andthe expression, within nerve cells, of at least one nucleic acidencoding for example a compound selected from a growth factor such asFGF, a trophic factor such as GDNF, BDNF, NGF, NT-3, a cytokine, acolony stimulating factor, an anticancer agent, a toxin, an enzyme, aneurotransmitter or a precursor thereof, a component of theextracellular matrix (ECM) such as N-CAM, PAS-NCAM, laminin,fibronectin, N-cadherin, a growth associated protein such as GAP-43,CAP-23 etc., enhancing the activity of the at least one functionalnucleic acid molecule capable of mediating RNA interference alsoproduced, and/or enhancing the prophylactic or therapeutic effectthereof.

Herein provided is a method for modulating, preferably repressingexpression of a target gene. Such a method may be used for preventing,treating or alleviating a nervous system disorder, in particular acentral nervous system (CNS) disorder, in an animal subject, inparticular a mammal, preferably a human, comprising administering tosaid animal (i) a pharmaceutical composition comprising a nonreplicative lentivirus comprising a lentiviral genome comprising anucleic acid sequence producing at least one functional nucleic acidmolecule capable of mediating RNA interference, preferably at least onefunctional miRNA, at least one functional shRNA, or at least onefunctional siRNA derived from said shRNA, said shRNA being designed tosilence the expression of a gene that encodes a protein of the astrocytecytoskeleton, said lentivirus being preferably pseudotyped for theselective transfer of the lentiviral genome into nervous cells, inparticular cells of the central nervous system, preferably glial cells,even more preferably astrocytes, and (ii) a pharmaceutically acceptablecarrier or excipient.

In a particular embodiment, invention also relates to a method asdescribed previously, wherein said method comprises two steps consistingin successively contacting a cell with a lentivirus according to thepresent invention or a composition comprising such a lentivirus, andwith a modulating factor such as tetracycline, as previously described,and wherein said two steps may be inverted.

The target gene expression repression can be reversed upon withdrawal ofthe modulating factor or upon interruption of the modulating factortreatment or on the contrary upon administration, adjunction orapplication of a modulating factor, depending, as explained previously,on the transactivator used. Such a method can be realized in a dose- andtime-dependent manner.

The nervous system disorder is preferably a central nervous systemdisorder. Such a SNC disorder may be a brain or spinal cord trauma or astroke.

The disorder may also be any condition associated with the formation ofa glial scar, for example a brain or spinal cord trauma or stroke, or aneurodegenerative disease, including, but not limited to Parkinson'sdisease, Huntington's disease, Alzheimer's disease, Amyotrophic LateralSclerosis (ALS), Spinal Muscular Atrophy (SMA), multiple sclerosis,temporal lobe epilepsy, lupus erythematosus and human immunodeficiencyvirus dementia.

The doses of vector may be adjusted by the skilled person depending onthe route of administration, tissue, vector, compound, etc.

The composition is advantageously administered at a rate of about 0.01to 10⁴ ng of P24 capsidic protein preferably between about 5 to 400 ngof capsidic protein P24. The lentiviruses may be purified andconditioned in any suitable composition, solution or buffer, comprisingpharmaceutically acceptable an excipient, vehicle or carrier, such as asaline, isotonic, buffered solution such as Mannitol 20%, optionallycombined with stabilizing agents such as isogenic albumin or any otherstabilizing protein, glycerol, etc., and also adjuvants such aspolybrene or DEAE dextrans, etc.

Various protocols may be used for the administration, such assimultaneous or sequential administration, single or repeatedadministration, etc., which may be adjusted by the skilled person.

A lentiviral vector can be used that provide for transient expression ofsiRNA molecules in the case of non integrative lentiviral vectors. Suchvectors can be repeatedly administered as necessary.

The pharmaceutical composition containing the lentivirus according tothe invention may be administered to a patient intracerebrally,intraspinally or systemically given the particular tropism of thepseudotyped lentiviral vectors, in particular for nervous cells such asglial cells of the astrocyte type.

Thus, it may be an administration given intracerebrally, intraspinally,i.e., directly in the medullar parenchyma, intra-striatally,intra-venously, intra-arterially. Preferred modes of injection areintracerebral injection, intraspinal injection, intrathécale injection.

Also herein provided is a kit for expressing a nucleic acid as hereindescribed, designed to silence the expression of a gene encoding aprotein of the astrocyte cytoskeleton, comprising (i) at least one nonreplicative lentivirus according to the present invention comprising alentiviral genome comprising a nucleic acid sequence producing saidnucleic acid designed to silence the expression of a gene, saidlentivirus being pseudotyped for the selective transfer of thelentiviral genome into cells of the central nervous system, andoptionally (ii) a leaflet providing guidelines.

Also provided is a cloning kit comprising:

a) a vector plasmid comprising a sequence, as described previously, suchas LTR-psi-Promoter-transgene-LTR(ΔU3),b) a trans-complementing plasmid comprising a sequenceCMV-Δpsi-gag-pol-Δenv-PolyA,c) an envelope plasmid comprising a sequence CMV-env-PolyA, the envelopebeing preferably an envelope of the rabies virus serogroup, andoptionallyd) a leaflet providing guidelines.

Further aspects and advantages of this invention are disclosed in thefollowing experimental section, which should be regarded as illustrativeand not limiting the scope of this application in any way.

Experimental Part

1. Development of Lentiviral Vectors that Allow Inhibition of GFAP andVimentin Expression.

a) Description of the Lv-shGFAP and Lv-shVIM Vectors:

In order to drive a powerful and long-term inhibition of GFAP andVimentin expression in reactive astrocytes, inventors developedlentiviral vectors that express shRNAs directed against GFAP andVimentin.

Inventors screened the cDNA encoding murine GFAP and vimentine todeterminate oligonucleotides sequences that are capable of efficientlysuppressing the expression of both proteins. Using a plasmid vector,candidate sequences were expressed as short hairpin RNAs (shRNAs) in HEK293 T cells cotransfected with a plasmid expressing the fusion proteinGFAP-GFP (or Vimentin-GFP). Efficient shRNAs caused the destruction ofthe unique GFAP-GFP mRNA (or Vimentin-GFP) and yielded thereby a declinein GFP fluorescence as was followed by Fluorescence Activated CellSorting (FACS). Results of the screening are represented on FIG. 3.

Two shRNAs were obtained that decreased the expression of GFAP-GFP by90%. The shGFAP sequence SEQ ID NO: 1 matches with mouse genome, and theshGFAP sequence SEQ ID NO: 2 matches with mouse, rat and human genome.Moreover two shRNAs were obtained that decreased the expression ofVimentin-GFP by 60% (SEQ ID NO 3 and 4 match with murine genome). Thesesequences match with mouse genome. Human siVIM sequence has beendescribed by Harborth et al. (2001).

To express these shRNAs in primary cultured astrocytes and in vivo,inventors then constructed lentiviral vectors that deliver shRNA againstGFAP or vimentine (these vectors were respectively called Lv-shGFAP andLv-shVIM). These vectors are derived from the lentivirus HIV-1. SelectedshRNA sequence was inserted downstream from the human U6 promoter intoprecursor plasmid <<pFlap>>. The vectors derived from this precursorplasmid contain the central HIV-1 Flap sequence, which facilitatetransduction of non-dividing cells (Zennou et al., 2001). Furthermore,the Woodchuck Hepatitis Virus Responsive Element (WPRE, Zufferey et al.,1999) was included into this precursor plasmid to protect the RNA genomeof the vector from RNAi mediated degradation during the production ofthe vector particles. Using this WPRE element, several teams haveproduced high-titred stocks of lentiviral vectors expressing shRNAs(Rubinson et al. 2003, Tiscornia et al. 2003). For safety reasons the U3promoter region is deleted from the 3′ LTR so that the vector is nonreplicative (Zufferey et al., 1998). In order to control transductionefficiency in vitro and in vivo, inventors also inserted the codingsequence of the fluorescent protein E-GFP, downstream from theubiquitous PGK promoter.

Lentivirus vector particles were produced by transient co-transfectionof HEK 293-T cells by the precursor plasmid, an encapsidation plasmid(p8.9) and an envelope expression plasmid. Lentiviruses produced by thismethod could be pseudotyped by different kind of envelopes. Lv-shGFAPand Lv-shVIM were pseudotyped with VSV envelope, which allow ubiquitouscell transduction, or with Mokola envelope, which target glial cells invivo (Mammeri et al., in preparation).

The structure of these vectors are described in FIG. 4.

b) In Vitro Characterization of the Lentiviral Vectors:

Before their use in vivo, the lentiviral vectors Lv-shGFAP and Lv-shVIMwere first tested in three different in vitro models, in order toevaluate their ability to reduce GFAP and vimentine expression, tomodulate astrocytic response to a lesion, and to promote neuronalsurvival and neurite growth.

In a first model of primary cultured astrocytes derived from the brainsof newborn mice, the ability of the lentiviral vectors Lv-shGFAP andLv-shVIM to mediate silencing of endogenous GFAP and Vimentin wasevaluated. These cultures are good models of reactive astrocytes(Bignami and Dahl, 1989; Privat et al., 1995). The cells were transducedwith different quantities of the vectors, and the expression of GFAP andvimentine has been monitorised by Western Blot analysis. Two weeks aftertransduction, an up to 90% reduction of the expression of the GFAP andvimentine was obtained in comparison with controls, as revealed in FIG.5.

The lentiviral vectors Lv-shGFAP and Lv-shVIM were then applied into anastrocytes-neurons coculture model, in order to specify whether thesevectors affects neuronal survival and/or neurite outgrowth. In thismodel, cortical neurons were prepared from 14 days old mice embryos andwere cultured on neonatal astrocytes previously transduced withLv-shGFAP, Lv-shVIM and different control vectors (which are representedby the following vectors: Lv-PGK-GFP, Lv-shRANDOM and Lv-shG1). Afterone week of coculture, the cells were fixed and βIII-tubulinimmunostaining was performed in order to detect the neuronal pericaryonsand neurites. Inventors demonstrate that the lentiviral vectorLv-shGFAP, alone or associated with the vector Lv-shVIM, induce asignificant increase in neuronal survival and in neurite outgrowth (seeFIG. 6). These results establish the ability of the Lv-shGFAP lentiviralvector to promote neuronal survival/axonal regrowth in an in vitro modelof glial reactivity.

In a third model of in vitro scratch wound, inventors evaluated theresponse of reactive astrocytes, transduced with the Lv-shGFAP andLv-shVIM vectors. In this model astrocytic monolayers, previouslytransduced with the different lentiviral vectors, were scratched with asterile pipette tip. After 2 days, one week and 2 weeks of incubation,cells were fixed and GFP immunostaining was performed in order tovisualize the transduced cells. In comparison to controls, cellstransduced with Lv-shGFAP and Lv-shVIM fail to repair the scratch woundat the different times of fixation. These results show that Lv-shGFAPand Lv-shVIM lentiviral induce a modulation of the astrogliosisphenotype and a reduction in the astrocytic scarring process in vitro.

2. Application of the Lentiviral Vectors Allowing RNAi-MediatedInhibition of GFAP And Vimentin in CNS Pathologies. a) Spinal CordInjury

In order to promote axonal regeneration and functional recovery afteracute traumatic injury, inventors developed a therapeutic strategy basedon the injections of Lv-shGFAP and Lv-shVIM lentiviral vectors into anin vivo model of spinal cord injury. This model consists in completeunilateral hemisection of the spinal cord in adult C57BL/6 mice. Inthese animals, they injected directly Lv-shGFAP and Lv-shVIM lentiviralvectors in the medullar parenchyma. They first developed an injectionprocedure in order to transduce a maximal number of reactive astrocytesaround the lesion area, in both rostro-caudal and dorso-ventral axis.More specifically they determined injection parameters that allowtransduction of precise spinal cord areas which are covered by neuronaltracts implicated in locomotion control, such as the corticospinal tractor the serotonergic fibers in the ventral horn. These injectionparameters are (i) 4 injection sites as described in FIG. 7, (ii) 2injections sub-sites that refer to two different injection depth: 1 mmand 0.5 mm (iii) a volume of 1 μl per site, (iv) an injection speed of0.2 μl per minute (v) a dose of 100 ng P24 of lentiviral vector persite, (vi) intraperitoneal administration of mannitol 20% 15 minutesbefore vectors injection.

Technically, the hemisection is performed at thoracic level T12, inorder to promote axonal regrowth upstream from the Central PatternGenerator (CPG), which is located at lumbar level L2-L3 in the mouse.The injection procedure comprises 4 injections sites around the lesion.In each injection site, the lentiviral vectors are applied along 2sub-sites, which correspond to two different depth levels of injection(respectively 0.5 and 1 mm). Lentiviral vectors are injected dorsally,in the vertical axis, by using tapered glass capillaries, with avelocity of 0.2 microliters per minute. The total volume of injectedlentiviral vectors was 1 μl per site, and the total amount of vectorinjected per site was 500 000 viral particles (˜100 ng P24). MoreoverMannitol 20% was administrated in the animal by intraperitonealinjection 15 minutes before the lentiviral vectors injection in order toincrease the transduction efficiency, as it was previously described foradenoviral vectors (Ghodsi et al., 1999).

This injection procedure allows the transduction of a large area aroundthe lesion site which can be extended on 500 μm in the dorso-ventralaxis. Inventors were able to block the GFAP and Vimentin expression in alarge region around the lesion, which lead to a significantly reducedglial reactivity.

Inventors then confirmed in vivo the inhibition of the endogenoussurexpression of GFAP and Vimentin by the Lv-shGFAP and Lv-shVIM vectorsafter injury. As presented in FIG. 8, GFAP expression is decreased inthe spinal cord area transduced with the lv-shGFAP lentiviral vector. Aspresented in FIG. 9, Vimentin is decreased in the spinal cord with thelv-shVIM lentiviral vector. These results show clearly the ability ofthe vectors Lv-shGFAP and Lv-shVIM to reduce the glial scar in vivo Infurther studies different series of hemisectioned mice were treated withdifferent lentiviral vectors including the Lv-shGFAP and Lv-shVIMvectors, and with control vectors. In these animals inventors analysethe formation of the glial scar, the axonal regeneration around thelesion site and finally the functional recovery, using the same methodsand behavioural tests used to characterize the double mutant (GFAP −/−,vim −/−) mice (Menet et al., 2003).

The functional recovery of the treated animals was evaluated by thebehavioural test named grid walk. As illustrated in FIG. 10, in thistest, the animals treated with the Lv-shGFAP, the Lv-shVIM, or bothvectors, present the best scores of recuperation, when compared tocontrols. The recuperation score is significantly increased for animalstreated with the Lv-shGFAP vector or with both Lv-shGFAP and Lv-shVIMvectors when datas are analyzed with the statistical Mann-Whitney test(*p<0.05) Additionally, an automated analysis of locomotion wasperformed before and one month after the surgery, on the same animals.Moreover in the same animals on which the behavioural test wasperformed, inventors also monitored immunohistochemical analyses inorder to detect axonal regeneration of serotonergic fibres in theventral horn, that are implicated in locomotion. The results show thatventral horns of animals treated with the Lv-shGFAP and Lv-shVIM containmore serotonergic fibers than control animals. (see FIG. 11)

These results of in vivo application show that the Lv-shGFAP vector,alone or associated with the Lv-shVIM vector, allow sustained reductionof GFAP expression in vivo and promote functional recovery after spinalcord lesion

b) Parkinson Disease

Inventors applied the Vimentin and GFAP KO approach to a degenerativepathology, namely Parkinson disease.

For that purpose, control mice as well as mice knocked out (KO) forGFAP, Vimentin or both genes were injected with the toxin6-hydroxy-dopamine (6-OH-DA) in the striatum, in order to induce apartial degeneration of dopaminergic neurons of the substantia nigra.Mice were then blindly followed for one month with a battery offunctional tests and then sacrificed to analyze the anatomical substrateand in particular the possible regeneration of dopaminergic axons.

In a study performed on a group of six animals comprising GFAP KO mice,double GFAP/Vimentin KO mice or control mice, inventors evaluated thesurvival of dopaminergic neurons after 6-OH-DA lesion of the substantianigra.

GFAP KO and double GFAP/vimentine KO mice present no decrease of thedopaminergic neurons number or a significant increase reaching up to 60%of the dopaminergic neurons number, in the injured side compared to thenon-injured side. On the contrary control mice present a significantdecrease of 40 to 60% of the dopaminergic neuron number, in the injuredside compared to the non-injured side. These results show an increasedplasticity in the GFAP KO mice and in the double GFAP/vimentin KO micewhich is related to a permissive glial substrate. The demonstration thatLv-shGFAP and Lv-shVIM can modulate glial permissivity in vitro and invivo, seriously suggests that these vectors can reduce the dopaminergicneuron loss in animal models of Parkinson disease.

REFERENCES

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1. A non replicative lentivirus comprising a lentiviral genomecomprising a nucleic acid sequence producing at least one functionalmicro RNA (miRNA), at least one functional short-hairpin RNA (shRNA)and/or at least one functional short interfering RNA (siRNA), saidmiRNA, shRNA and siRNA being designed to silence the expression of agene that encodes a protein of the astrocyte cytoskeleton, saidlentivirus being pseudotyped for the selective transfer of thelentiviral genome into cells of the central nervous system.
 2. Thelentivirus according to claim 1, wherein the lentiviral genome furthercomprises a second nucleic acid sequence producing at least onefunctional miRNA, at least one functional shRNA and/or at least onefunctional siRNA, said miRNA, shRNA and siRNA being designed to silencethe expression of a gene that encodes a different protein of theastrocyte cytoskeleton.
 3. The lentivirus according to claim 1, whereinthe protein of the astrocyte cytoskeleton is selected from GFAP andvimentin.
 4. The lentivirus according to claim 3, wherein, when theshRNA is designed to silence a gene encoding GFAP, said shRNA is: (SEQID NO: 1) (i) ACCGAGAGAGATTCGCACTCAATATTCAAGAGATATTGAGTGCGAATCTCTCTCTTTTTATCGATG, or (SEQ ID NO: 2) (ii)ACCGAGATCGCCACCTACAGGAAATTCAAGAGATTTCCTGTAGGTGGC GATCTCTTTTTATCGATG,

and, wherein, when the shRNA is designed to silence a gene encodingvimentin, said shRNA is: (SEQ ID NO: 7) (i)ACCGAATGGTACAAGTCCAGGTTTGTTCAAGAGACAAACTTGGACTTG TACCATTCTTTTTCTCGAGG,or (SEQ ID NO: 8) (ii) ACCGAGAGAAATTGCAGGAGGAGATTCAAGAGATCTCCTCCTGCAATTTCTCTCTTTTTCTCGAGG.


5. The lentivirus according to claim 3, wherein, when the siRNA isdesigned to silence a gene encoding GFAP, said siRNA is: (i)GAGAGAGATTCGCACTCAATA, (SEQ ID NO: 3) (ii) TATTGAGTGCGAATCTCTCTC, (SEQID NO: 4) (iii) GAGATCGCCACCTACAGGAAA (SEQ ID NO: 5) or (iv)TTTCCTGTAGGTGGCGATCTC, (SEQ ID NO: 6)

and, wherein, when the siRNA is designed to silence a gene encodingvimentin, said siRNA is: (i) GAATGGTACAAGTCCAGGTTTG, (SEQ ID NO: 9) (ii)CAAACTTGGACTTGTACCATTC, (SEQ ID NO: 10) (iii) GAGAGAAATTGCAGGAGGAGA (SEQID NO: 11) or (iv) TCTCCTCCTGCAATTTCTCTC. (SEQ ID NO: 12)


6. The lentivirus according to claim 1, wherein the lentivirus isselected from the group consisting of Human Immunodeficiency Virus type1 (HIV-1), Human Immunodeficiency Virus type 2 (HIV-2), SimianImmunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV),Equine Infectious Anaemia Virus (EIAV), Bovine Immunodeficiency Virus(BIV), Visna Virus of sheep (VISNA) and Caprine Arthritis-EncephalitisVirus (CAEV).
 7. The lentivirus according to claim 1, wherein thelentivirus is deprived of any lentiviral coding sequence and of theenhancer region of the U3 region of the LTR3′.
 8. The lentivirusaccording to claim 1, wherein said lentivirus is pseudotyped with alyssavirus envelope, in particular with a virus envelop of the rabiesvirus serogroup selected from the group consisting of Rabies (RAB);Duvenhague (DUV), European Bat type 1 (EB-1), European Bat type 2(EB-2), Kotonkan (KOT), Lagos Bat (LB), Mokola (MOK), Obodhiang (OBD)and Rochambeau (RBU), or any chimeric composition of these envelopes. 9.The lentivirus according to claim 1, wherein said lentivirus ispseudotyped with an alphavirus envelope, in particular with a virusenvelop of the Ross River Virus (RRV).
 10. The lentivirus according toclaim 1, wherein said lentivirus is pseudotyped with an areanviridaeenvelope, in particular with a virus envelop of the Lymphocyticchoriomeningitis virus (LCMV).
 11. The lentivirus according to claim 1,wherein said lentivirus is pseudotyped with a vesiculovirus envelope.12. The lentivirus according to claim 1, wherein the lentiviral genomecomprises, between LTR3′ and LTR5′ sequences, a lentiviral ψencapsidation sequence, a coding sequence producing shRNA, andoptionally a promoter, a sequence enhancing RNA nuclear import, asequence enhancing RNA nuclear export, a transcription regulationelement, and/or a mutated integrase.
 13. The lentivirus according toclaim 12, wherein the promoter is a viral promoter or a cellularpromoter.
 14. The lentivirus according to claim 13, wherein the promoteris a cellular promoter allowing expression of a shRNA, selected from thegroup consisting of U6, H1 and 7SK RNA polymerase III promoter.
 15. Thelentivirus according to claim 13, wherein the promoter is a viralpromoter allowing expression of a miRNA, selected from the groupconsisting of CMV, TK, RSV LTR polymerase II promoter.
 16. Thelentivirus according to claim 13, wherein the promoter is a cellularpromoter allowing expression of a miRNA, selected from the groupconsisting of PGK, Rho, EF1α, GFAP, Vimentin, Nestin, S100β polymeraseII promoter.
 17. The lentivirus according to claim 12, wherein thepromoter is a transactivator induced promoter that modulates RNAinterference, said transactivator induced promoter comprising aplurality of transactivator binding sequences operatively linked to thenucleic acid sequence producing shRNA.
 18. The lentivirus according toclaim 17, wherein the transactivator induced promoter is atetracycline-dependent transactivator selected from the rtTA-Oct.2transactivator composed of the DNA binding domain of rtTA2-M2 and of theOct-2^(Q)(Q→A) activation domain, and the rtTA-Oct.3 transactivatorcomposed of the DNA binding domain of the E. coli Tet-repressor proteinand of the Oct-2^(Q)(Q→A) activation domain.
 19. The lentivirusaccording to claim 12, wherein the sequence enhancing the ARN nuclearimport is lentiviral cPPT CTS (Flap) sequence.
 20. The lentivirusaccording to claim 12, wherein the sequence enhancing the ARN nuclearexport comprises HIV-1 REV response element (RRE) sequence.
 21. Thelentivirus according to claim 12, wherein the sequence enhancing thenuclear export comprises the CTE element
 22. The lentivirus according toclaim 12, wherein the transcription regulation element is selected fromWoodchuck hepatitis virus responsive element (WPRE), APP UTR5′ region,TAU UTR3′, and insulators MAR, SAR, S/MAR, scs and scs' sequence. 23.The lentivirus according to claim 12, wherein the integrase comprises amutation in at least one of its basic region and/or catalytic regionresponsible for the lentivirus to be non integrative.
 24. A method forpreventing or treating a central nervous system (CNS) disorder in ananimal, comprising administering to said animal a pharmaceuticalcomposition comprising a non replicative lentivirus comprising alentiviral genome comprising a nucleic acid sequence producing at leastone functional miRNA, at least one functional shRNA and/or at least onefunctional siRNA, said miRNA, shRNA and siRNA being designed to silencethe expression of a gene that encodes a protein of the astrocytecytoskeleton, said lentivirus being pseudotyped for the selectivetransfer of the lentiviral genome into cells of the central nervoussystem, and a pharmaceutically acceptable excipient.
 25. The methodaccording to claim 24, wherein said animal is a human.
 26. The methodaccording to claim 24, wherein the disorder is a brain or spinal cordtrauma or a stroke.
 27. The method according to claim 24, wherein thedisorder is a neurodegenerative disease selected from Parkinson'sdisease, Huntington's chorea, Alzheimer's disease, Amyotrophic LateralSclerosis (ALS) and Spinal Muscular Atrophy (SMA).
 28. The methodaccording to claim 24, wherein the administration of the pharmaceuticalcomposition is by intracerebral, intraspinal or intrathecal injection.29. A kit for expressing a nucleic acid designed to silence theexpression of a gene encoding a protein of the astrocyte cytoskeleton,comprising at least one non replicative lentivirus comprising alentiviral genome comprising a nucleic acid sequence producing at leastone functional miRNA, at least one functional shRNA and/or at least onefunctional siRNA, said miRNA, shRNA and siRNA being designed to silencethe expression of a gene that encodes a protein of the astrocytecytoskeleton, said lentivirus being pseudotyped for the selectivetransfer of the lentiviral genome into cells of the central nervoussystem.